大鼠血管内皮生长因子shRNA表达质粒的构建及鉴定

Construction and identification of VEGF shRNA eukaryotic expression plasmid in rat

目的:构建大鼠血管内皮生长因子(Vascular endothelial growth factor,VEGF)短发夹RNA(Short hairpin RNA,shRNA)真核表达质粒,为利用RNA干扰技术(RNA interference,RNAi)从转录后水平抑制角膜新生血管的研究做准备。方法:用DNA重组技术根据大鼠VEGF mRNA序列设计并合成3条shRNA寡核苷酸片段,退火形成双链并克隆进入载体pSilencer2.0 U6中,进行酶切鉴定和测序。结果:酶切证明构建的shRNA序列已插入载体,经测序证明与设计的相同,获得大鼠VEGF shRNA表达质粒。结论:成功构建了3条大鼠VEGF shRNA真核表达质粒,为靶向VEGF的RNAi抑制角膜新生血管奠定了基础。

Objective： To construct rat vascular endothelial growth factor short hairpin RNA eukaryotic plasmid expression vector, in order to provide a basis for inhibition of corneal neovascularization by RNA interference. Methods ： Three different shRNAs targeting the coding sequence of the rat VEGF mRNA were designed. After annealing the formed double stranded DNAs were inserted into the plasmid pSilencer2.0 U6. Then the recombinant plasmids were identified by enzyme digestion and DNA sequencing. Results： The three VEGF shRNAs were successfully inserted into the plasmid vector, and the coding sequences of the obtained shRNAs were consistent with the designed fragments. Conclusion： The recombinant plasmids of VEGF shRNAs were successfully constructed, which will be useful to inhibit corneal neovascularization.